In the production of viral vectors for use in gene therapy replication-deficient viruses are used for reasons of safety. Replication-deficient viruses are able to infect a human cell in vivo and transfer the transgene into the cell, but are unable to replicate in the cell by themselves. Typically, this is achieved by deleting viral genes which are important for virus replication, such as for example rep and cap genes. This also allows for incorporation of gene product(s) of interest in place of the viral genes. For the generation of virus particles in a host cell, the viral genes that are deleted from the replication-deficient virus need to be provided separately, for instance by providing helper viruses.
Adeno-associated virus (AAV) is a non-autonomously replicating virus that belongs to the Parvoviridae family and constitutes a single-stranded molecule of DNA with an outer icosahedral coat of structural protein having a diameter of approx. 18 to 26 nm. Wild type AAV viruses can either integrate into the genome of the host cell or replicate in the host cells in the presence of a helper virus. Adenoviruses were first identified as possible helper viruses. However, other essentially spherically shaped mammalian helper viruses, such as the herpes viruses, which are pathogenic to humans and animals, are also suitable. In recent years, adeno-associated virus (AAV) vector production by means of baculovirus-based expression systems in insect cells (Urabe et al. [2002] Hum Gene Ther. 13(16):1935-43) has become increasingly popular since the system is easily scalable for industrial applications of gene therapy. In this production system typically three recombinant baculoviruses are used encoding the AAV rep genes, the AAV cap genes and the gene product of interest (transgene DNA) flanked by AAV inverted terminal repeats (ITRs).
A significant disadvantage associated with the preparation of viral vectors using helper viruses or virus-based expression systems is the formation of a mixed population of product virus particles and helper viruses, which has to be subjected to further purification. Contaminating viruses, such as for example adenovirus or baculovirus, have to be avoided or minimized when using viral vectors in gene therapy because of the potential pathogenicity and/or immunogenicity of the contaminating virus.
Several methods for elimination of helper viruses are presently known in the art, however, they all have their disadvantages. Examples of these methods are density gradient centrifugation or heat inactivation or a combination thereof. However, density gradient centrifugation is only economically feasible on relatively small volumes and thus this method is not feasible on an industrial scale. Heat inactivation is based on the different thermal stabilities of AAV and the helper viruses. For example, heating a mixed population of adenovirus and AAV to 56-65° C. leads to more or less selective heat inactivation of the helper virus with only a slight loss in the activity of the AAV. Unfortunately, the denatured helper virus proteins which would be still present in the sample and upon use in gene therapy be able to evoke a cellular immune response in the patient (Smith, C. A. et al. (1996) J. Virol., 70, 6733-6740).
In addition, column chromatographic methods, including ion exchange (anion and/or cation based) and affinity chromatography, have been developed to purify AAV vectors. Those methods result in highly enriched preparations of AAV vector, but they cannot be validated alone to show sufficient depletion of helper viruses to meet the regulatory requirements of marketed pharmaceutical products.
Finally, in U.S. Pat. No. 6,479,273 it is disclosed that separation of recombinant AAV and adenovirus, i.e., two essentially spherical viruses of substantially different diameter is achieved by subjecting a solution containing both viruses to one or more filtrations through a filter membrane with a pore size of approximately 50 nm or a filter membrane with a pore size of approximately 35 nm rAAV is disclosed to have a diameter of approx. 25 nm and adenovirus is said to have a diameter of approx. 65-90 nm, although larger diameters, e.g. close to 100 nm, have been reported in literature [Kennedy and Parks (2009) Molecular Therapy 17(10):1664-1666; Berkowitz (2003) WCBP 7th annual meeting Jan. 7-10, 2003, San Francisco, Calif.]. However, contaminating viruses that result from AAV vector production by means of a baculovirus-based expression system in insect cells, are derived from the baculoviridae and thus are rod-shaped, with a length of approximately 260 nm and a diameter of approximately 20 nm. Since (recombinant) AAV is substantially spherical and has a diameter of approximately 18 to 26 nm, the baculovirus partially has (in two dimensions) a similar size to the target virus.
Regulatory requirements, for example the viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (ICH Q5A (R1)) by the European Medicines Agency (EMA), require that the process for the purification of a biological pharmaceutical is capable of removing any non-product virus. The removal of viral contaminants is performed by “viral clearance” or “viral removal” process steps and is usually obtained by chromatography and/or virus filtration. Also “virus inactivation” process steps are used to attenuate potential pathogenic effects caused by non-product viruses. This usually contains extreme physical conditions (e.g. pH, Temperature) and/or chemical conditions (e.g., detergents, solvents). Pharmaceutical products are usually proteins of less than approximately 200 kDa and “virus removal” processes are well established. However, a relatively new type of products concern gene therapy products that comprise viruses of a few thousand kDa for which “virus removal” processes are not well documented. In particular, a process of “virus removal” in which the pharmaceutical product is a spherical virus and the viral contaminant is a rod-shaped virus is not documented yet.
Therefore, there is a need in the art for additional separation/purification methods that are technically and economically feasible to be employed on industrial scale and that are capable of partly or completely removing a non-product virus of a virus-based expression system, from a sample containing AAV, preferably a recombinant AAV sample obtained from a baculovirus-based expression system. Thereby reducing the potential pathogenicity and/or immunogenicity of the non-product virus. In particular there is a need in the art for additional separation/purification methods where the non-product virus is rod-like shaped that has a length of several times the diameter of AAV, but has a similar diameter as the AAV.